Mechanical loading of the skeleton is a powerful stimulus for new bone formation (osteogenesis). Bone formation in many contexts (e.g., development, fracture healing) depends critically on the formation of new blood vessels (angiogenesis), yet the role of angiogenesis in supporting mechanical loading-induced osteogenesis is not known. Deficits in vascularity due to aging and diabetes may compromise the skeletal response to mechanical stimuli and thus may contribute to osteoporotic fracture risk. Using the rat forelimb loading model, we recently quantified a rapid increase in vascularity after damaging fatigue loading of bone, prior to woven bone formation. We also quantified the upregulation of angiogenic genes (e.g., VEGF, vascular endothelial growth factor) and the osteoinductive gene BMP-2 (bone morphogenetic protein-2) within 1 hr after fatigue loading, with the unexpected finding that BMP-2 expression occurred first in vascular cells then in bone cells. Functionally, woven bone formation occurred in proportion to the level of fatigue damage and whole-bone strength was restored two weeks after loading - regardless of the level of initial damage. Notably, angiogenic inhibition markedly reduced the woven bone response after fatigue loading. A fundamental unknown remains - what are the biological factors that cause woven bone formation after fatigue loading versus lamellar bone formation after anabolic loading? In the next project period we will use the in vivo loading model of forelimb compression (in rats and mice) as we work toward the long term goal of determining the mechano-biological pathways that lead to rapid non-endochondral bone formation after mechanical loading. In Aim 1, we will compare molecular, vascular and bone formation responses to fatigue loading versus anabolic loading, and determine the role of developmental osteogenesis (in particular Hedgehog signaling) and angiogenesis in loading-induced woven and lamellar bone formation. In Aim 2, we will determine if BMP signaling (and in particular endothelial BMP-2) is required for loading-induced osteogenesis and angiogenesis. Taken together, these studies will establish important mechano-biological factors that mediate the distinct processes of lamellar versus woven bone formation and clarify the role of angiogenesis. They will extend our understanding of the osteogenic response to skeletal stress injuries (fatigue fractures) and may serve as a basis for future development of strategies for rapid bone formation to augment weak or damaged bones. PUBLIC HEALTH RELEVANCE: The skeleton is regulated in part by mechanical forces. The goal of this project is to understand the biological pathways that control bone formation following mechanical stimulation. This information may lead to future strategies to increase bone mass and reduce skeletal fragility.